Viruses with an RNA genome replicate and evolve via mechanisms distinct from DNA genomes and are important human, animal and plant pathogens. The mammalian immune mechanisms that defend against RNA viruses are poorly characterized. The proposed research will genetically dissect the immune pathway of Caenorhabditis elegans animals triggered by Flock house virus (FHV), one of the best characterized RNA viruses. We have shown recently that FHV replication launched from an inducible transgene integrated in the C. elegans genome triggers production of virus-derived siRNAs, which guide viral RNA clearance by RNA silencing or RNA interference (RNAi). Further genetic analyses have provided evidence for a conserved RNA silencing virus immunity (RSI) pathway in C. elegans in response to either FHV or another distinct RNA virus. We have carried out pilot feeding RNAi and chemical mutagenesis screens using the C. elegans/FHV system. Our feeding RNAi screen has identified 35 genes required for RSI including drh-1, which is dispensable for RNAi induced by exogenous dsRNA and highly homologous to the recently identified mammalian immune receptor for RNA viruses. Viable RSI-defective worm mutants recovered from the chemical screen include both RNAi-defective and RNAi-sensitive mutants and none are allelic with drh-1 or another frequent occurring RNAi-defective allele. Hence, our results have shown that C. elegans RSI requires components that are shared with, and dispensable for, exogenous RNAi, as well as those homologous to mammalian viral immunity components. We plan to further characterize the mechanism of viral RNA clearance guided by viral siRNAs, the features and roles of secondary viral siRNAs, and the specificity mechanism of RSI components dispensable for exogenous RNAi in worms. Further, we will isolate additional components of the animal RSI pathway through genome-wide feeding RNAi and chemical mutagenesis screens using the methodology already established in the lab. The proposed experiments will provide the first genome-view of the viral immunity pathway in whole animals, identify novel RNAi components that cannot be obtained in previous screens using non-viral RNAi triggers, and reveal immune mechanisms against RNA viruses that facilitate understanding mammalian viral immunity. PUBLIC HEALTH RELEVANCE: The nematode C. elegans has been a powerful model for elucidating the molecular mechanisms of both RNA interference (RNAi) and programmed cell death in humans. Many RNA viruses such as influenza, hepatitis C virus, poliovirus, dengue virus and West Nile virus are important human pathogens. Thus, it is likely that the proposed studies on the RNAi-mediated viral immunity pathway of C. elegans to RNA viruses will provide mechanistic insights into mammalian viral immunity and RNAi.